Human polymorphonuclear leukocytes (PMN) are at the front-line of cellular innate immune-mediated host defense against infection, especially those caused by organisms such as S. aureus (SA). Despite the potent PMN antimicrobial system, 10-20% of ingested SA remained viable, but not replicating, in PMN. Persistence of SA within PMN has profound clinical consequences, as signature features of staphylococcal infection are relapse, metastases, and failure of antibiotics to which SA are susceptible. In our ongoing studies of the fate of SA-laden human PMN and their interactions with monocyte-derived macrophages (M), we have made several novel findings: PMN-SA (1) early after phagocytosis of SA display a phenotype that is atypical for pathogen-induced apoptosis; (2) are not efficiently efferocytosed by M; (3) maintain sustained levels of proliferating cell nuclear antigen (PCNA), a cell-cycle protein expressed in PMN cytoplasm, and recently linked to prolonging PMN survival by scavenging procaspases; and (4) lyse in a fashion most consistent with necroptosis, a caspase-independent programmed necrotic cell death pathway never previously described in PMN but involving a multicomponent cytoplasmic signaling complex and dependence on receptor-interacting protein kinase 1 (RIP1K). We reason that these cellular events in and between PMN-SA and M drive the clinical hallmarks of SA infection and propose studies to explore of the mechanisms underlying our novel observations. Aim 1: Determine the mechanisms underlying the failed efferocytosis of PMN-SA by M? 1A: Identify signals generated by PMN-SA that promote efferocytosis by M 1B: Identify signals generated by PMN-SA that actively block efferocytosis by M 1C: Identify signaling pathways and effector responses of M challenged with PMN-SA 1D: Determine the role of ectosomes generated by PMN-SA to modulate efferocytosis by M Aim 2: Identify cellular components, biochemical events, and signaling pathways underlying necroptosis of PMN-SA 2A: Determine the composition and regulation of the ripoptosome in PMN-SA 2B: Determine the contribution of PCNA to the initial prolonged survival and eventual lysis of PMN-SA 2C: Determine the signaling pathways engaged as PMN-SA proceed to necroptosis 2D: Determine the role of TNF?-dependent signaling in necroptosis of PMN-SA Our studies will identify mechanisms responsible for the atypical apoptosis, failed efferocytosis, and necrotic death of PMN-SA, thereby providing novel insights into fundamental aspects of phagocyte control of the inflammatory response and new potential therapeutic targets.